Electrospray ionization (ESI) mass spectrometry1, 2 has rapidly become an important tool in the field of structural biochemistry. The technique allows folded proteins to be ionized, sometimes with evidence for little change in gross three-dimensional structure. The resulting ions can then be studied in the gas phase using the tools of modern mass spectrometry.3-8 Not only can single proteins be studied using this methodology, but multi-protein and protein-ligand complexes sometimes can also be ionized intact, although the number of thoroughly studied examples is much smaller. Recently, ionization of such complex structures as a whole ribosome9 has been demonstrated. Protein complexes in the gas phase can be studied by tandem or multiple-stage mass spectrometry.10-12 In such procedures, the original complex can be made to undergo successive dissociation processes, revealing the molecular weights of the individual constituents. Unlike most other techniques, mass spectrometry is not restricted to the detection of certain types of constituents of a molecular complex, such as those labeled with fluorophores or otherwise made visible to the analytical method.
Proteins and other biologically relevant macromolecular systems usually show one or a small number of conformations under physiological conditions, a feature essential for playing a well-defined biochemical role. The solution phase structure is generally assumed to be different from the most stable conformation in the gas phase.3, 4, 9, 13-15 The main requirement for developing successful mass spectrometric techniques is therefore to preserve these metastable solution structures and this demands minimizing the internal energy of the ions in order to keep the gas-phase unfolding or dissociation rates as low as possible. This task is generally performed by avoiding denaturing conditions when the solution is prepared for mass spectrometry and adjusting pressure and lens potential values carefully in the source and atmospheric interface region of the instrument.10, 16 The key aim in these procedures is to desolvate protein ions and to direct them into the high-vacuum region of a mass spectrometer without affecting the non-covalent interactions that maintain the highly ordered structures. This objective is usually achieved by applying relatively high pressures in the atmospheric interface and low potential gradients throughout the lens system16. High gas pressures provide high collision frequencies in the first vacuum region of the instrument, which keeps the ions at low temperatures via collisional cooling and also facilitates efficient desolvation. However, since both the solvent envelope and ion conformation are maintained by non-covalent interactions, there is often a compromise between conditions that preserve the intact structure and those needed for complete desolvation. Furthermore, the instrumental settings that allow gentle desolvation are usually not optimal for ion transfer efficiency, so the sensitivity of the instrument can be seriously degraded.
Nanospray17, 18 is often the ionization method of choice to achieve gentle desolvation while also providing a high ionization efficiency for small, valuable samples. Unlike traditional commercially available ESI ion sources,18 nanospray is compatible with aqueous buffers at physiological pH and its sample consumption is one or two orders of magnitude lower due to the high ionization efficiency. High ionization efficiency and efficient desolvation are characteristics usually attributed to the low solution flow rate that is known to reduce the size of the charged droplets initially produced. The smaller initial droplets undergo fewer coulomb-fissions and each evaporates less solvent, which results in lower concentrations of non-volatile matrix components in the final nanodroplet that yields the actual gaseous protein ion. Smaller initial droplet sizes also accelerate ion formation and in this way a higher portion of the droplets will actually be completely desolvated to provide ions that are available for mass analysis. Nanospray is generally assumed to provide better desolvation efficiency than ESI. This feature is attributed to more efficient solvent evaporation from the smaller droplets and lower solvent vapor load on the atmospheric interface due to considerably lower sample flow rates. The intrinsically good desolvation efficiency does not require the application of harsh desolvation conditions in the atmospheric interface (high temperature, high cone voltage, etc.), which in turn enhances the survival of fragile biochemical entities including non-covalent complexes. In spite of these advantages, nanospray mass spectra depend strongly on the nanospray tip used; the tip-to-tip reproducibility of spectra is weak. Furthermore, tip geometry may change due to arcing or break during operation. Another difficulty with nanospray is the lack of control over the spray process: in practice the spray cannot be adjusted, it can only be turned on and off by changing the high voltage.19, 20 High flow rates and extremes of pH are generally required.
Both in the case of nanospray and conventional forced-flow, pneumatically assisted electrospray, the absolute sensitivity is influenced not only by the width in m/z units of individual peaks, but by the shape and width of the overall charge state distribution. The shapes of charge state distributions are frequently used as a diagnostic tool for determining the degree of unfolding of proteins in the course of ionization.21-26 Broad charge state distributions at high charge states are generally associated with unfolded structures, while narrow distributions at lower charge states are treated as diagnostic of native or native-like folded ion structures in the gas phase. A model developed recently by Kebarle et al. evaluates the maximum number of charges of protein ions based on the relative apparent gas phase basicities (GB) of possible charge sites on the protein molecule.26-29 This model describes protein ion formation from buffered solutions in electrospray via the formation of proton-bound complexes with buffer molecules at each charge site and the subsequent dissociation of these complexes. The branching ratios for dissociation of these complexes depend on the relative apparent GB of the buffer molecule (e.g. ammonia in the case of ammonium buffers) relative to that of the protein charge site. Apparent GB values of particular sites on proteins can be estimated based on the intrinsic GB values of chemical moieties, the electric permittivity of the protein molecule and the spatial distribution of charges, which latter factor is related to the size of the protein ion. The observed charge state distribution is a result of these factors, the temperature of desolvation and any further charge reduction as a result of ion/molecule reactions occurring in the atmospheric interface or during passage through the ion optics of the mass spectrometer.
In principle, the spray process and charging of the sample can be decoupled and the originally charged liquid can initially be finely dispersed by a different spraying technique. This approach is widely implemented in commercial ESI sources by means of pneumatic spraying,30 often in order to roughly disperse the large amounts of liquid sample coming from a standard liquid chromatograph. Since d ˜1/vg2 where d is the mean diameter of droplets, vg is the linear velocity of the nebulizing gas at high linear gas velocities and high gas/liquid mass flow ratios, droplet sizes comparable to nanospray can be achieved theoretically.31 
Although complete ionization of complex sample materials, such as proteins, that are supplied in an aqueous solution buffered to a physiological pH has been achieved to some degree in the reduced atmosphere of a mass spectrometer capable of sampling at atmospheric pressure, gaseous ionization of samples to yield substantially a single species for each component of the solution when the material is a protein in an aqueous solution buffered to physiological pH has not been known previously. Careful investigation of ESI has determined that, in fact, ionized liquid droplets are produced by prior art methods. The ionized liquid is sampled and evaporation is completed in the mass spectrometer after the droplets have been heated and sometimes subjected to multiple collisions, resulting in some unfolding of protein samples, which leads to an undesirably broad charge distribution. Complete gaseous ionization of a sample material from a solution outside a mass spectrometer has not previously been accomplished although progress in this direction is being made by the method of laser-assisted spray ionization.32 